A rapid protocol for inducing acute pancreatitis in zebrafish models.

Acute pancreatitis (AP) is an inflammatory disorder that occurs in the exocrine pancreas associated with tissue injury and necrosis. Experimental models of AP typically involve rodents, such as rats or mice. However, rodents exhibit divergent pathophysiological responses after the establishment of AP between themselves and in comparison, with human. The experiments conducted for this manuscript aimed to standardize a new AP model in zebrafish and validate it. Here, we provide a protocol for inducing AP in zebrafish through intraperitoneal injections of synthetic caerulein. Details are provided for solution preparation, pre-injection procedures, injection technique, and monitoring animal survival. Subsequently, validation was performed through biochemical and histological analyses of pancreatic tissue. The administered dose of caerulein for AP induction was 10 µg/kg applied four times in the intraperitoneal region. The histological validation study demonstrated the presence of necrosis within the first 12 h post-injection, accompanied by an excess of zymogen granules in the extracellular milieu. These observations align with those reported in conventional rodent models. We have standardized and validated the AP model in zebrafish. This model can contribute to preclinical and clinical studies of new drugs for AP treatment. Therefore, this novel model expands the toolkit for exploring faster and more effective preventive and therapeutic strategies for AP.

The tissue-specific nature of physiological zebrafish mitochondrial bioenergetics.

Zebrafish are a powerful tool to study a myriad of experimental conditions, including mitochondrial bioenergetics. Considering that mitochondria are different in many aspects depending on the tissue evaluated, in the zebrafish model there is still a lack of this investigation. Especially for juvenile zebrafish. In the present study, we examined whether different tissues from zebrafish juveniles show mitochondrial density- and tissue-specificity comparing brain, liver, heart, and skeletal muscle (SM). The liver and brain complex IV showed the highest O2 consumption of all ETC in all tissues (10x when compared to other respiratory complexes). The liver showed a higher potential for ROS generation. In this way, the brain and liver showed more susceptibility to O2- generation when compared to other tissues. Regarding Ca2+ transport, the brain showed greater capacity for Ca2+ uptake and the liver presented low Ca2+ uptake capacity. The liver and brain were more susceptible to producing NO. The enzymes SOD and Catalase showed high activity in the brain, whereas GPx showed higher activity in the liver and CS in the SM. TEM reveals, as expected, a physiological diverse mitochondrial morphology. The essential differences between zebrafish tissues investigated probably reflect how the mitochondria play a diverse role in systemic homeostasis. This feature may not be limited to normal metabolic functions but also to stress conditions. In summary, mitochondrial bioenergetics in zebrafish juvenile permeabilized tissues showed a tissue-specificity and a useful tool to investigate conditions of redox system imbalance, mainly in the liver and brain.

Using structural equation modeling to assess the genotoxic and mutagenic effects of heavy metal contamination in the freshwater ecosystems: A study involving Oreochromis niloticus in an urban river.

Chemical pollutants represent a leading problem for aquatic ecosystems, as they can induce genetic, biochemical, and physiological changes in the species of these ecosystems, thus compromising their adaptability and survival. The Capibaribe River runs through the state of Pernambuco, located in Northeastern Brazil, and passes through areas of agricultural cultivation, densely populated cities, and industrial centers, primarily textiles. Despite its importance, few ecotoxicological studies have been conducted on its environment, and knowledge about pollution patterns and their effects on its biota is still being determined. The objective of this study was to evaluate the water quality and the damage supposed to be caused by pollutants on the DNA specimens of Nile tilapia (Oreochromis niloticus) obtained from seven strategic points of Capibaribe. Tilapia specimens and water were collected during the rainy and dry seasons from 2015 to 2017. The following characteristics were analyzed: physicochemical (six), metal concentration (seven), local pluviosity, micronuclei, and comet assay. The physicochemical and heavy metal analyses were exploratory, whereas the ecotoxicological analyses were hypothetical. To verify this hypothesis, we compared the groups of fish collected to the results of the micronuclei test and comet assay. We created a Structural Equation Model (SEM) to determine how each metal's micronuclei variables, damage index, pluviosity, and concentration were related. Our results demonstrated that the highest values for markers of genetic damage were detected at points with the highest heavy metal concentrations, especially iron, zinc, manganese, chromium, and cadmium. The SEM demonstrated that metals could explain the findings of the genotoxicity markers. Moreover, other pollutants, such as pesticides, should be considered, mainly where the river passes through rural areas. The results presented here demonstrate that the Capibaribe River has different degrees of contamination and confirm our hypothesis.

Performance of collagen-based matrices from Nile tilapia skin: A pilot proteomic study in a murine model of wound healing.

Full-thickness cutaneous trauma, due to the lack of dermis, leads to difficulty in epithelialization by keratinocytes, developing a fibrotic scar, with less elasticity than the original skin, which may have disorders in predisposed individuals, resulting in hypertrophic scar and keloids. Biomedical materials have excellent characteristics, such as good biocompatibility and low immunogenicity, which can temporarily replace traditional materials used as primary dressings. In this work, we developed two dermal matrices based on Nile tilapia collagen, with (M_GAG) and without (M) glycosaminoglycans, using a sugarcane polymer membrane as a matrix support. To assess the molecular mechanisms driving wound healing, we performed qualitative proteomic analysis on the wound bed in an in vivo study involving immunocompetent murine models at 14 and 21 days post-full-thickness skin injury. Gene Ontology and Pathway analysis revealed that both skins were markedly represented by modulation of the immune system, emphasizing controlling the acute inflammation response at 14 and 21 days post-injury. Furthermore, both groups showed significant enrichment of pathways related to RNA and protein metabolism, suggesting an increase in protein synthesis required for tissue repair and proper wound closure. Other pathways, such as keratinization and vitamin D3 metabolism, were also enriched in the groups treated with M matrix. Finally, both matrices improved wound healing in a full post-thick skin lesion. However, our preliminary molecular data reveals that the collagen-mediated healing matrix lacking glycosaminoglycan (M) exhibited a phenotype more favorable to tissue repair, making it more suitable for use before skin grafts.

Shrimp laccase degrades polycyclic aromatic hydrocarbons from an oil spill disaster in Brazil: A tool for marine environmental bioremediation.

Our work aims to purify, characterize and evaluate a laccase from by-products of the shrimp farming industry (Litopenaeus vannamei) for the degradation of Polycyclic Aromatic Hydrocarbons (PAHs) from 2019 oil spill in Brazilian coast. The enzyme was purified by affinity chromatography and characterized as thermostable, with activity above 90 °C and at alkaline pH. In addition, the laccase was also tolerant to copper, lead, cadmium, zinc, arsenic, hexane and methanol, with significant enzymatic activation in acetone and 10 mM mercury. Concerning PAHs' degradation, the enzyme degraded 42.40 % of the total compounds, degrading >50 % of fluorene, C4-naphthalenes, C3-naphthalenes, C2-naphthalenes, anthracene, acenaphthene, 1-methylnaphthalene and 2-methylnaphthalene. Thus, this laccase demonstrated important characteristics for bioremediation of marine environments contaminated by crude oil spills, representing a viable and ecological alternative for these purposes.

Carboxymethylchitosan with medium-molecular-weight affects kinectics and acrosome of stallion sperm after freezing/thawing.

Semen cryopreservation ensures the storage of stallion genetics for an unlimited time. The improvement of extenders with new antioxidant substances can optimize the properties of post-thawed semen. The study aimed to investigate the addition effect of medium-molecular-weight carboxymethylchitosan (CQm) derivates to freezing diluent of stallion sperm after freezinf/thawing. Twice a week, five ejaculates of four stallions were obtained, totalizing 20 ejaculates. Semen was diluted in commercial freezing extender (Botucrio) supplemented with CQm: control (0), 0.75, 1.5, and 3 mg/mL. Samples were filled in straws (0.5 mL) and submitted to freezing and storage at -196°C. Thawing was performed at 37°C/30 s, and the samples of each group were analyzed for kinetics, plasma membrane integrity, acrosome membrane integrity, and mitochondrial membrane potential . The addition of 1.5 and 3 mg/mL CQm showed lower values (P < .05) of total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP) and wobble (WOB), comparing to control group. Besides, it was observed lower (P < .05) percentages of sperm with intact acrosomes in the group treated with 3 mg/mL of CQm than control group. In conclusion, high concentration of medium-molecular-weight carboxymethylchitosan to freezing diluent damages kinematic and acrosome of stallion sperm after freezing/thawing.

A circular approach for the efficient recovery of astaxanthin from Haematococcus pluvialis biomass harvested by flocculation and water reusability.

Flocculation has been proved an efficient method for microalgal biomass harvesting, but some coagulant agents may have adverse effects on microalgae growth, making the reuse of the medium unfeasible. In this study, Haematococcus pluvialis was harvested by different flocculants, and the feasibility of the reuse of the culture medium was evaluated. Results suggested that both inorganics, polyaluminum chloride (PA) and ferric chloride (FC), and organics, extracted from Moringa oleifera seed (MSE) and chitosan (CH) resulted in efficient flocculation - flocculation efficiency above 99 %. However, using PA and FC had adverse effects on the astaxanthin recovery from haematocysts - losses of 58.6 and 73.5 %, respectively. Bioflocculants in the reused medium also had higher growth performance than inorganic ones. Furthermore, bioflocculants in reused medium increase the contents of ß-carotene, astaxanthin, and linolenic acid. This investigation demonstrated that using MSE and CHI for harvesting H. pluvialis enables the water reusability from a flocculated medium.

Biocompatibility and immunostimulatory properties of fish collagen and shrimp chitosan towards peripheral blood mononuclear cells (PBMCs).

Several naturally occurring biopolymers are commercially produced from livestock and farmed animals processing wastes, including aquatic organisms. These wastes are considered valuable coproducts of fishery processing industry, from which biopolymers may be recovered and exploited for their bioactive potential. The aim of this work was to prepare polymeric films from collagen and chitosan solutions, extracted from fishery discards, and investigate the cytotoxicity and immunomodulatory activity towards human peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from healthy donors and treated with Chitosan, Collagen, Chitosan+Collagen solutions and Chitosan+Collagen film in order to measure the changes in cell viability, cytosolic calcium concentration ([Ca2+]cyt), mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS) levels, differentiation and activation of T CD8+ and CD4+ lymphocytes, and cytokine production. Results showed that collagen and chitosan preparations did not show cytotoxic effect, while cellular IL-6, IL-10, and TNF-α release was observed. Chitosan and collagen were able to promote non-cytotoxic PBMCs activation through cytosolic and mitochondrial ROS production. There was a noteworthy phenotyping of lymphocytes T CD8+ and CD4+ counting and an increase of [Ca2+] cyt and ΔΨm levels. These results suggest that chitosan/collagen-based biomaterials produce immunostimulatory effects on PBMC with potential to biomedical approaches.

Acetylcholinesterase from the charru mussel Mytella charruana: kinetic characterization, physicochemical properties and potential as in vitro biomarker in environmental monitoring of mollusk extraction areas.

Acetylcholinesterase (AChE; EC 3.1.1.7) from aquatic organisms have been used to evaluate the exposure of specimens to pesticides and heavy metals at sublethal levels in environmental samples. AChE of Mytella charruana was extracted to characterize its physicochemical and kinetic properties as well as the effect of organophosphate (dichlorvos, diazinon, chlorpyrifos, methyl-parathion and temephos), carbamates (carbaryl, carbofuran and aldicarb), benzoylureas (diflubenzuron and novaluron), pyrethroid (cypermethrin) and juvenile hormone analog - JHA (pyriproxyfen) and the effect of metal ions: Hg2+, Cd2+, Pb2+, As3+, Cu2+ and Zn2+, in order to evaluate the potential of the enzyme as biomarker. The optimum pH of M. charruana AChE was 8.5 and the maximum activity peak occurred at 48 °C, being highly thermostable maintaining 97.8% of its activity after incubation at 60 °C. The Michaelis-Menten constants (km) for the substrates acetylthiocholine and propionylthiocholine were 2.8 ± 1.26 and 4.94 ± 6.9 mmol·L-1, respectively. The Vmax values for the same substrates were 22.6 ± 0.90 and 10.2 ± 4.94 mU·mg-1, respectively. Specific inhibition results suggest an AChE presenting active site with dimensions between those of AChE and butyrylcholinesterase (BChE). The IC20 values related to the effect of the pesticides on the enzyme showed higher inhibitory power of temephos (0.17 µmol·L-1), followed by aldicarb (0.19 µmol·L-1) and diflubenzuron (0.23 µmol·L-1). Metal ions inhibited M. charruana enzyme in the following order: Hg2+ > Pb2+ > Cd2+ > As3+ > Cu2+ > Zn2+. These data suggest that the enzyme showed potential as in vitro biomarker of the exposure to temephos, mercury, zinc and copper.

Binding Mechanism between Acetylcholinesterase and Drugs Pazopanib and Lapatinib: Biochemical and Biophysical Studies.

Tyrosine kinase inhibitors (TKIs) are antitumor compounds that prevent the phosphorylation of proteins in a biological environment. However, the multitarget performance of TKIs promotes them as possible candidates for drug repositioning. In this work, interaction and inhibition studies through spectroscopic and computational techniques to evaluate the binding effectiveness of lapatinib and pazopanib TKIs to acetylcholinesterase (AChE) are reported. The results indicated potent inhibition at the µM level. The types of inhibition were identified, with pazopanib acting through non-competitive inhibition and lapatinib through acompetitive inhibition. The fluorescence suppression studies indicate a static mechanism for lapatinib-AChE and pazopanib-AChE systems, with a binding constant in the order of 105 M-1. The obtained thermodynamic parameters reveal interactions driven by van der Waals forces and hydrogen bonds in the lapatinib-AChE system (ΔH° and ΔS° < 0). In contrast, the pazopanib-AChE system shows positive ΔH° and ΔS°, characteristic of hydrophobic interactions. The Foster resonance energy transfer study supports the fluorescence studies performed. The 3D fluorescence studies suggest changes in the microenvironment of the tryptophan and tyrosine residues of the protein in contact with lapatinib and pazopanib. The results suggest effective inhibition and moderate interaction of the drugs with AChE, making them interesting for conducting more in-depth repositioning studies as AChE inhibitors.

Non-clinical repeated dose 28-day oral toxicity, reproductive toxicity and cytotoxicity studies of the polar fraction of Parkinsonia aculeata aerial parts extract.

This study was aimed to evaluate toxicity in repeated doses for 28 days, reproductive toxicity and cytotoxicity of a polar fraction obtained from the hydroethanolic extract of Parkinsonia aculeata (PfrHEPA) in experimental models. To perform the toxicity test in repeated doses for 28 days, male and female Wistar rats were treated via orogastric for 28 days with PfrHEPA (35, 70 or 140 mg/kg) according to the guidelines established by the Organisation for Economic Co-operation and Development (OECD) number 407 (1995). For assessment, the impact of PfrHEPA on the reproductive output various parameters were measured, including maternal weight, no. of pregnant females, female fertility index (%), gestation lengthtime, implantation sites, litter size and placental index of test animals. The cytotoxicity of PfrHEPA was performed on the tumor lines NCI-H292 (human lung carcinoma), HL-60 (human promyelocytic leukemia) and HCT-116 (colorectal cancer). In the repeated dose toxicity test for 28 days, no mortality was observed in the male and female rats treated with PfrHEPA as well as morphological changes and biochemical and hematological parameters. In the reproductive toxicity test, no abnormalities were observed related to the toxicological parameters in both mothers and offspring. Regarding the cytotoxicity assay, the PfrHEPA fraction did not demonstrate significant cytotoxic effect on the cell lines analyzed. The present results suggest the use of PfrHEPA is safe and well tolerated in rats. Further studies are planned to identify and purify the active compounds for subsequent in vivo evaluation.

Chlorella vulgaris functional alcoholic beverage: Effect on propagation of cortical spreading depression and functional properties.

Recent advances in microalgae biotechnology have proven that these microorganisms contain a number of bioactive molecules, that can be used as food additives that help prevent disease. The green microalga Chlorella vulgaris presents several biomolecules, such as lutein and astaxanthin, with antioxidant capacity, which can play a protective role in tissues. In this study, we produced and analyzed a C. vulgaris functional alcoholic beverage (produced using a traditional Brazilian alcoholic beverage, cachaça, and C. vulgaris biomass). Assays were conducted in vitro by radical scavenging tests, and in vivo, by modeling cortical spreading depression in rat brains. Scavenging radical assays showed that consumption of the C. vulgaris alcoholic beverage had a DPPH inhibition of 77.2%. This functional alcoholic beverage at a concentration of 12.5 g L-1 significantly improved cortical spreading depression velocity in the rat brains (2.89 mm min-1), when compared with cachaça alone (3.68 mm min-1) and control (distilled water; 3.25 mm min-1). Moreover, animals that consumed the functional beverage gained less weight than those that consumed just alcohol and the control groups. These findings suggest that the C. vulgaris functional alcoholic beverage plays a protective physiologic role in protecting brain cells from the effects of drinking ethanol.

A highly stable raw starch digesting α-amylase from Nile tilapia (Oreochromis niloticus) viscera.

A raw starch digesting α-amylase from Nile tilapia (Oreochromis niloticus) intestine was identified. The α-amylase, AMY-T, had an estimated molecular weight of 60 kDa and purified to near homogeneity. AMY-T showed an apparent KM 4.78 mg/mL and Vmax 0.44 mg/mL/min) towards soluble starch. It was highly stable for 24 h in the pH range 3.0-10.0, and to solvents like methanol, isopropanol, butanol, dimethylformamide, DMSO and ethyl-ether. AMY-T was able to digest different carbohydrates, mainly showing endo-activity. Importantly, AMY-T was catalytically efficient and adsorbing towards raw potato starch at temperature documented for other raw starch digesting α-amylases. Thin layer and anion exchange chromatography characterization showed that the end products of raw starch hydrolysis were glucose, maltose and maltodextrins, with degree of polymerisation ranging 1-8. Scanning electron microscopy analysis of the AMY-T treated starch granules documented both granular exo- and endo-attack by AMY-T. These catalytic capabilities suggest high potential for AMY-T for industrial use.

Impact on cholinesterase-inhibition and in silico investigations of sesquiterpenoids from Amazonian Siparuna guianensis Aubl.

The plant popularly known as "negramina" (Siparuna guianensis Aubl.), member of the family Siparunaceae produces an essential oil that presents several biological activities reported in literature. Here, the essential oil was obtained by hydrodistillation from fresh leaves collected in the state of Roraima, far north of the Amazon. Chemical composition of the essential oil was characterized by gas chromatography coupled to mass spectrometry (GC-MS) and flame ionization detector (GC-FID). The sesquiterpenoid shyobunone and its derivatives were identified as major compounds in the oil (>40%). The effect of S. guianensis essential oil on the acetylcholinesterase (AChE) activity from Crassostrea rhizophorae, Litopenaeus vannamei and Electrophorus electricus was tested by spectrophotometric assays. The essential oil has been identified as an AChE inhibitor. The mechanism of inhibition was investigated as well as spectrofluorimetric interactions between the essential oil and the enzyme. 1H NMR titration and molecular docking were also investigated. The spectrophotometric results revealed that shyobunone and its derivatives strongly interact with AChE with a kind of non-competitive inhibition. Interaction studies support the results of enzyme inhibition. Molecular coupling predicted that iso-shyobunone is the strongest ligand, corroborated by fluorescence suppression and 1H NMR titration results. In conclusion, Siparuna guianensis essential oil can be a new source of shyobunone and derivatives capable to reversibly inhibit AChE showing potential neuroprotective properties to be applied in the treatment of Alzheimer's disease.

Effects of pyriproxyfen on zebrafish brain mitochondria and acetylcholinesterase.

Pyriproxyfen is an insecticide used worldwide that acts as a biomimetic of juvenile hormone. This study investigated metabolic and synaptic impairments triggered by pyriproxyfen using zebrafish acetylcholinesterase (zbAChE) and mitochondria as markers. A brain zbAChE assay was performed in vitro and in vivo covering a range of pyriproxyfen concentrations (0.001-10 µmol/L) to assess inhibition kinetics. Docking simulations were performed to characterize inhibitory interactions. Zebrafish male adults were acutely exposed to 0.001, 0.01 and 0.1 µg/mL pyriproxyfen for 16 h. Mitochondrial respiration of brain tissues was assessed. ROS generation was estimated using H2DCF-DA and MitoSOX. Calcium transport was monitored by Calcium Green™ 5 N. NO synthesis activity was estimated using DAF-FM-DA. Brain acetylcholinesterase showed an in vivo IC20 of 0.30 µmol/L pyriproxyfen, and an IC50 of 92.5 µmol/L. The inhibitory effect on zbAChE activity was competitive-like. Respiratory control of Complex I/II decreased significantly after insecticide exposure. The MitoSOX test showed that O2- generation had a pyriproxyfen dose-dependent effect. Brain tissue lost 50% of Ca2+ uptake capacity at 0.1 µg/mL pyriproxyfen. Ca2+ release showed a clear mitochondrial impairment at lower pyriproxyfen exposures. Thus, Ca2+ transport imbalance caused by pyriproxyfen may be a novel deleterious mechanism of action. Overall, the results showed that pyriproxyfen can compromise multiple and interconnected pathways: (1) zbAChE impairment and (2) the functioning of the electron transport chain, ROS generation and calcium homeostasis in zebrafish brain mitochondria. Considering the many similarities between zebrafish and human, more caution is needed when pyriproxyfen is used in both urban and agricultural pest control.

Mitochondria as targets for toxicity and metabolism research using zebrafish.

BACKGROUND: The study of mitochondrial functions in zebrafish was initiated before the 1990s and has effectively supported many of the recent scientific advances in the functional studies of mitochondria. SCOPE OF REVIEW: This work elaborates various peculiarities and general advances in the study of mitochondria using this animal model. MAJOR CONCLUSIONS: The inclusion of zebrafish models in scientific research was initiated with structural studies of mitochondria. Then, toxicological studies involving chemical compounds were undertaken. Currently, there is a decisive tendency to use zebrafish to understand how chemicals impair mitochondrial bioenergetics. Zebrafish modeling has been fruitful for the analysis of ion homeostasis, especially for Ca2+ transport, since zebrafish and mammals have the same set of Ca2+ transporters and mitochondrial membrane microdomains. Based on zebrafish embryo studies, our understanding of ROS generation has also led to new insights. GENERAL SIGNIFICANCE: For the study of mitochondria, a new era was begun with the inclusion of zebrafish in bioenergetics research.

Brain acetylcholinesterase of three perciformes: From the characterization to the in vitro effect of metal ions and pesticides.

Estuarine environments gather pollution from large regions including urban and industrial zones. The monitoring of environmental quality in these areas constitutes a real requirement for global sustainable development. Therefore, the aim of this study was to characterize the physicochemical and kinetic parameters of brain acetylcholinesterase (AChE) in the species Centropomus undecimalis, Diapterus auratus and Diapterus rhombeus and to assess the effects (in vitro) of pesticides and metal ions on their respective activities in order to investigate them as potential biomarkers. Physicochemical properties such as thermostability, optimal pH and temperature, as well as kinetic parameters were investigated. AChE was pointed as the predominant cholinesterase (ChE) in the brains of the species under study. The highest optimum pH value was observed for C. undecimalis (8.0), and the lowest for D. rhombeus and D. auratus, with 7.2 and 7.0, respectively. The optimal temperature was 35 °C for the three species. The AChEs of the three species presented moderate thermostability, since they retained 61%, 72% and 67% of the activity up to 45 °C (C. undecimalis, D. auratus and D. rhombeus, respectively). The carbamate carbofuran showed to be the strongest inhibitor even at very low concentrations (IC50: 0.182, 0.174 and 0.203 µmol/L - C. undecimalis, D. auratus and D. rhombeus, respectively), followed by dichlorvos and carbaryl. According to the findings, the AChE of these species may be proposed as in vitro biomarker of exposure to carbofuran and dichlorvos (all three species) and carbaryl (D. auratus and D. Rhombeus), as well as for exceeding limit concentrations of Hg2+ (D. rhombeus) and As3+ (D. auratus) in biomonitoring programs located or not at estuarine environments.

Evaluation of antioxidant and antibacterial capacity of green microalgae Scenedesmus subspicatus.

Microalgae are considered one of the most promising raw materials for the development of high value products for pharmaceuticals, nutraceuticals, and cosmetic industries, as well as being potential sources of protein, vitamins, and minerals for human consumption. Hence, the present research focuses extraction of antioxidant and antimicrobial compounds from Scenedesmus subspicatus using solvents of different polarities. Different solvents such as ethanol, methanol, butanol, acetone, dimethyl sulfoxide, and water were used to extract compounds from the green microalgae S. subspicatus and then they were examined for phytochemical screening, antioxidant activity, and antimicrobial properties. In vitro free radical quenching and total antioxidant activity of extracts were investigated with 1,1-diphenyl-2-picryl hydrazyl and compared with catequin and gallic acid as positive controls. The antimicrobial activity was evaluated in gram-negative and gram-positive bacteria. Aqueous extracts and dimethyl sulfoxide presented better performance in phytochemical analysis. This result showed consistency in the sequential tests. The antioxidant activity was also better using the two solvents cited above. The extracts acetone, water, and dimethyl sulfoxide showed ability to inhibit the growth of Bacillus subtilis. However, only dimethyl sulfoxide inhibited the growth of Klebsiella pneumoniae and Escherichia coli. Use of the aqueous extract, proven its effectiveness, is an economic protocol and avoids the use of toxic substances.

Effects of dietary Bacillus subtilis and Shewanella algae in expression profile of immune-related genes from hemolymph of Litopenaeus vannamei challenged with Vibrio parahaemolyticus.

B. subtilis and S. algae effects in growth, survival and innate immunity were assessed on L. vannamei juveniles. During 60 days, shrimp were reared in three treatments: Bs, fed with 106 CFU of B. subtilis per gram of commercial feed, Sa, fed with 106 CFU of S. algae per gram of commercial feed and Control (without bacterial addition). Then, the animals were subjected to a V. parahaemolyticus challenge. For this purpose, four treatments were established: Control (shrimp not submitted to probiotic treatments), Vibrio (Vibrio challenged shrimp), Vibrio + Bs (Bs challenged shrimp) and Vibrio + Sa (Sa challenged shrimp). Shrimp hemolymph was sampled 45-days after rearing and 24 h post-challenge for quantification of prophenoloxidase (proPO), lipopolysaccharide and ß-1,3-glucan-binding protein (LGBP) and hemocyanin (HEM) transcripts by qPCR. Moreover, shrimp final weight and survival were also verified. B. subtilis administration enhanced shrimp growth and improved proPO, LGBP and HEM expression levels before and after challenge. After 60-days of feeding, Sa final weight was higher than the Control, whereas Vibrio + Sa cumulative mortality after 48 h of Vibrio challenge was lower than Vibrio group. These results could be correlated with the proPO and LGBP up regulation in Vibrio + Sa compared to Vibrio group, protecting L. vannamei from the bacterial infection. Together, these results suggest the probiotic potential of B. subtilis e S. algae in the modulation of immune-related genes as a tool to control V. parahaemolyticus infection inside shrimp.

Characterization of brain acetylcholinesterase of bentonic fish Hoplosternum littorale: Perspectives of application in pesticides and metal ions biomonitoring.

Acetylcholinesterase (AChE; EC 3.1.1.7) is a serine hydrolase, whose main function is to modulate neurotransmission at cholinergic synapses. It is, therefore, the primary target of some pesticides and heavy metals. Its inhibition in aquatic organisms has been used as an indicator of the presence of these pollutants in water bodies. The present study aimed to characterize physicochemical and kinetic parameters of brain AChE in the benthic fish Hoplosternum littorale and to analyze the in vitro effects of pesticides (dichlorvos, diazinon, chlorpyrifos, parathion-methyl, temephos, carbaryl, carbofuran, aldicarb, diflubenzuron, novaluron and pyriproxyfen) and metal ions (As3+, Cd2+, Cu2+, Fe2+, Mn2+, Mg2+, K+, Pb2+, Hg2+, Zn2+) investigating the potential of this enzyme as environmental biomarker based on current regulations. Specific substrates and inhibitors have indicated AChE to be the predominant cholinesterase (ChE) in the brain of H. littorale. Peak activity was observed at pH 8.0 and 30 °C. The enzymatic activity is otherwise moderately thermostable (≈ 50% activity at 45 °C). The enzyme can reduce the activation energy of acetylthiocholine hydrolysis reaction to 8.34 kcal mol-1 while reaching a rate enhancement of 106. Among the pesticides under study, dichlorvos presented an IC50 value below the maximum concentrations allowed by legislation. This study presents the first report on the inhibition of brain AChE activity from Siluriformes by the pesticides novaluron and pyriproxyfen. Mercury ion also exerted a strong inhibitory effect on its enzymatic activity. The H. littorale enzyme thus has the potential to function as an in vitro biomarker for the presence of the pesticide dichlorvos as well as mercury in areas of mining and industrial discharge.